This application claims priority to German priority document 101 14 999.9, filed Mar. 26, 2001, that is hereby incorporated by reference.
The computer-readable sequence(s) on the attached compact disk are hereby incorporated by reference.
1. Field of the Invention
D-carbamoylases and polypeptides having D-carbamoylase activity, especially those from Arthrobacter. D-carbamoylases that are more economical, efficient and conveniently used in commercial and industrical processes, such as those with superior activity or stability. Nucleic acids, vectors and host cells encoding or expressing these D-carbamoylases. Methods for making an enantiomerically enriched or purified amino acid using such a D-carbamoylase and methods for identifying and isolating a gene, gene cluster or operon encoding a D-carbamoylase.
2. Description of Related Art
Carbamoylases are enzymes which are capable of converting N-carbamoylamino acids stereoselectively into the L- or D-amino acid, while retaining the enantiomeric carbamoylamino acid, see equation 1 in FIG. 10.
Racemic N-carbamoylamino acids can preferably be obtained quite easily from hydantoins by means of hydantoinases or by reaction of amino acids with KOCN, and for this reason such processes are used on an industrial scale for the preparation of enantiomerically concentrated amino acids (Drauz K, Kottenhahn M, Makryaleas K, Klenk H, Bernd M, Angew Chem, (1991). Chemoenzymatic synthesis of D-xcfx89-ureidoaminoacids, 103, 704-706; See Equation 2 in FIG. 11.
D-Carbamoylases are known in the literature (Syldatk et al. in xe2x80x9cEnzymatic Catalysis in Organic Synthesisxe2x80x9d, eds.: Drauz, Waldmann, VCH, 1st and 2nd Ed.), but these mostly do not work very efficiently or are unstable (Syldatk C, Mxc3xcller R, Pietzsch M, Wagner F (1992). Biocatalytic production of amino acids and derivatives; eds.: Rozzell D, Wagner F, Hanser Publishers, Munich; 129-176; Louwrier A, Knowles C. J. (1996). The purification and characterization of a novel D-specific carbamoylase enzyme from Agrobacterium sp. Enzyme Microb Technol. 19; 562-571; Nanba H, Ikenaka Y, Yamada Y, Yajima K, Takano M Takahashi S (1998). Isolation of Agrobacterium sp. strain KNK712 that produces N-carbamyl-D-amino acid amidohydrolase, cloning of the gene for this enzyme, and properties of the enzyme. Biosci. Biotechnol. Biochem. 62 (5) 875-881; Kim D. M., Kim G. J., Kim H. S. (1994). Biotechnol Lett, (16) 11-16). Accordingly, there is a need for further improved carbamoylases, such as those with improved stability or activity.
The present invention encompasses D-carbamoylases and polypeptides having D-carbamoylase activity, especially those from Arthrobacter. These D-carbamoylases provide a more economical, efficient and conveniently usable D-carbamoylase, for instance, a D-carbamoylase with superior stability or activity. The invention also encompasses nucleic acids encoding such a D-carbamoylases or polypeptide having a D-carbamoylase activity, as well as plasmids and microorganisms encompassing such a nucleic acid sequence. Methods for making an enantiomerically enriched or purified amino acid using such a D-carbamoylase and methods for identifying and isolating a gene, gene cluster or operon encoding a D-carbamoylase are also described.
The use of an enzymatic process for the synthesis of an organic compound, such as a D-amino acid is advantageous, particularly for large scale industrial process, since such a process often provides a superior product yield and improved reactant or product selectivity compared to a conventional chemical process. In nature, enzymatic process are of decisive importance, for instance, in the biosynthesis of proteins such as albumins. Accordingly, an efficient and convenient enzymatic process for producing enantiomerically concentrated amino acids is a preferred target of the present invention. Enantiomerically concentrated amino acids are important products for the synthesis of bioactive compounds or for the production of other products, such as those used for parenteral feeding.